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. 2007 Dec 26;2(12):e1354.
doi: 10.1371/journal.pone.0001354.

High levels of MeCP2 depress MHC class I expression in neuronal cells

Julie Miralvès  1 , Eddy Magdeleine, Lara Kaddoum, Hélène Brun, Sophie Peries, Etienne Joly
Affiliations

High levels of MeCP2 depress MHC class I expression in neuronal cells

Julie Miralvès et al. PLoS One. .

Abstract

Background: The expression of MHC class I genes is repressed in mature neurons. The molecular basis of this regulation is poorly understood, but the genes are particularly rich in CpG islands. MeCP2 is a transcriptional repressor that binds to methylated CpG dinucleotides; mutations in this protein also cause the neurodevelopmental disease called Rett syndrome. Because MHC class I molecules play a role in neuronal connectivity, we hypothesised that MeCP2 might repress MHC class I expression in the CNS and that this might play a role in the pathology of Rett syndrome.

Methodology: We show here that transiently transfected cells expressing high levels of MeCP2 specifically downregulate cell-surface expression of MHC class I molecules in the neuronal cell line N2A and they prevent the induction of MHC class I expression in response to interferon in these cells, supporting our first hypothesis. Surprisingly, however, overexpression of the mutated forms of MeCP2 that cause Rett syndrome had a similar effect on MHC class I expression as the wild-type protein. Immunohistological analyses of brain slices from MECP2 knockout mice (the MeCP2(tm1.1Bird) strain) demonstrated a small but reproducible increase in MHC class I when compared to their wild type littermates, but we found no difference in MHC class I expression in primary cultures of mixed glial cells (mainly neurons and astrocytes) from the knockout and wild-type mice.

Conclusion: These data suggest that high levels of MeCP2, such as those found in mature neurons, may contribute to the repression of MHC expression, but we find no evidence that MeCP2 regulation of MHC class I is important for the pathogenesis of Rett syndrome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MeCP2 overexpression diminishes MHC class I expression.
N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (Kk, Ld or Dd), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2A (grey fill) and 12 independent transfections with vectors expressing human MeCP2A (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2A or B isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, p<0.01 ; ***, p<0.001).
Figure 2
Figure 2. Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.
N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule Ld or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, p<0.05; **, p<0.01; ***, p<0.001).
Figure 3
Figure 3. Mutant forms of MeCP2 that cause RTT retain their repressive effect on MHC class I expression.
Panel A: Schematic representation of the MeCP2 protein. Red and orange arrows indicate the positions of the mutations introduced in MeCP2 by site-directed mutagenesis (MBD: methyl-CpG binding domain, TRD: transcription repression domain, WW: group II WW-domain-binding region). Panel B: N2A cells transfected with empty pcDNA3.1 (mock cells), with pcDNA3.1 expressing Myc-tagged MeCP2A (pMeCP2A-myc) or with pcDNA3.1 expressing Myc-tagged MeCP2A with the R133C point mutation (pMeCP2A-R133C-myc) were stained with mouse anti-Myc 9E10 monoclonal antibody, and FITC-labelled anti-mouse IgG antibody. Coverslips were mounted in DAPI-containing ProLong Gold antifade reagent (Molecular Probes) before observation by fluorescence microscopy. Panel C: N2A cells transfected as in panel B were double immunostained for cell surface MHC class I and intracellular Myc-tagged MeCP2, then analysed by flow cytometry. Similar data were obtained for all four mutated forms of MeCP2A and MeCP2B (not shown), and these observations were reproduced in three independent transfection experiments.
Figure 4
Figure 4. Evaluation of MHC class I expression in adult mouse brain slices.
Serial frozen sections of adult male wild-type and MeCP2−/y littermates were analysed for expression of MHC class I by immunohistochemistry using the rat R1-21.2 monoclonal antibody and EnVision detection technology (Dako). For the negative control, the same staining process was used omitting the primary antibody. Similar results were obtained with the M1/42 monoclonal antibody. Similar results were obtained in independent experiments on brains from three different pairs of mice.
Figure 5
Figure 5. Deletion of MECP2 does not affect basal or IFN-γ -induced MHC class I expression in primary cultures of mixed glial cells.
Mixed glial cell cultures established from two-day old wild-type, MeCP2+/− and MeCP2−/y mice (10, 6 and 8 animals per group, respectively) were treated or not with IFN-γ on the second day of culture and analysed two days later by flow cytometry for MHC class I expression. Neurons were identified by their intracellular staining with an anti-β-III-tubulin antibody (inset). Large cells, containing mainly astrocytes, were analysed separately by an appropriate forward/side scatter gate. Primary spleen fibroblasts from the same mice were also subjected or not to IFN-γ treatment and stained for their MHC class I expression. Panel A: Representative histograms showing cell surface staining (x axis) against cell number (y axis), obtained with cells from wild-type and MeCP2−/y male littermates. White-filled curves represent background staining, gray-filled curves represent MHC I-specific staining. Panel B: MHC class I fold-induction in response to IFN-γ was calculated as the ratio of MFI of treated cells (induced MHC I level) on MFI of untreated cells (basal MHC I level). Grey-filled squares show MHC class I fold-induction for individual mice and for each cell type. White-filled squares represent the group's mean of fold-inductions (±SD).
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